Differentiation of a CD4+/CD8αβ+ Double Positive T Cell Population From The CD8 Pool Is Sufficient To Mediate Graft-vs-Host Disease but not Graft-vs-Leukemia Effects

Acute graft-vs-host disease (aGVHD) and tumor relapse remain the primary complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT) for malignant blood disorders. While post-transplant cyclophosphamide has reduced the overall prevalence and severity of aGVHD, relapse rates remain a concern. Thus, there remains a need to identify the specific human T cell subsets mediating GVHD pathology versus graft-versus-leukemia (GVL) effects. In xenogeneic transplantation studies using primary human cells from a variety of donors and tissue sources, we observed the development of a mature CD4+/CD8αβ+ double positive T cell (DPT) population in mice succumbing to lethal aGVHD but not in mice that failed to develop aGVHD. The presence of DPT, irrespective of graft source, was predictive of lethal GVHD as early as one week after xenogeneic transplantation. DPT co-express the master transcription factors of the CD8 and CD4 lineages, RUNX3 and THPOK respectively, and produce both cytotoxic and modulatory cytokines. To identify the origin of DPT, we transplanted isolated human CD4 or CD8 T cells, which in turn revealed that DPT only arise from the CD8 pool. Interestingly, re-transplantation of sorted CD8 T cells from GVHD mice did not reveal a second wave of DPT differentiation. Re-transplantation of flow-sorted DPT, CD8 or CD4 T cells from GVHD mice revealed that DPT are sufficient to mediate GVHD pathology but not GVL effects versus B-cell acute lymphoblastic leukemia. Lastly, we confirmed the presence and correlation of DPT with aGVHD pathology in a small cohort of allo-HSCT patients that developed grade 2-4 aGVHD in our clinic. Further understanding of DPT differentiation and pathology may lead to targeted prophylaxis and/or treatment regimens for aGVHD and potentially other human chronic inflammatory diseases.

A human SIRPA knock-in xenograft mouse model to study human hematopoietic and cancer stem cells

In human-to-mouse xenogeneic transplantation, polymorphisms of signal-regulatory protein α (SIRPA) that decide their binding affinity for human CD47 are critical for engraftment efficiency of human cells. In this study, we generated a new C57BL/6.Rag2nullIl2rgnull (BRG) mouse line with Sirpahuman/human (BRGShuman) mice, in which mouse Sirpa was replaced by human SIRPA encompassing all 8 exons. Macrophages from C57BL/6 mice harboring Sirpahuman/human had a significantly stronger affinity for human CD47 than those harboring SirpaNOD/NOD and did not show detectable phagocytosis against human hematopoietic stem cells. In turn, Sirpahuman/human macrophages had a moderate affinity for mouse CD47, and BRGShuman mice did not exhibit the blood cytopenia that was seen in Sirpa−/− mice. In human to mouse xenograft experiments, BRGShuman mice showed significantly greater engraftment and maintenance of human hematopoiesis with a high level of myeloid reconstitution, as well as improved reconstitution in peripheral tissues, compared with BRG mice harboring SirpaNOD/NOD (BRGSNOD). BRGShuman mice also showed significantly enhanced engraftment and growth of acute myeloid leukemia and subcutaneously transplanted human colon cancer cells compared with BRGSNOD mice. BRGShuman mice should be a useful basic line for establishing a more authentic xenotransplantation model to study normal and malignant human stem cells.

Transcriptomic and Proteomic Analyses Reveal the Potential Mode of Action of Chondrocyte Sheets in Hyaline Cartilage Regeneration

Chondrocyte sheet transplantation is a novel and promising approach to treating patients who have cartilage defects associated with osteoarthritis. Hyaline cartilage regeneration by autologous chondrocyte sheets has already been demonstrated in clinical research. In this study, the efficacy of polydactyly-derived chondrocyte sheets (PD sheets) as an allogeneic alternative to standard chondrocyte sheets was examined using an orthotopic xenogeneic transplantation model. In addition, the expression of genes and the secreted proteins in the PD sheets was analyzed using a microarray and a DNA aptamer array. The efficacy of PD sheets with respect to cartilage defects was assessed using histological scores, after which the expressions of genes and proteins exhibiting a correlation to efficacy were identified. Enrichment analysis of efficacy-correlated genes and proteins showed that they were associated with extracellular matrices, skeletal development, and angiogenesis. Eight genes (ESM1, GREM1, SERPINA3, DKK1, MIA, NTN4, FABP3, and PDGFA) exhibited a positive correlation with the efficacy of PD sheets, and three genes (RARRES2, APOE, and PGF) showed a negative correlation for both transcriptomic and proteomic analyses. Among these, MIA, DKK1, and GREM1 involved in skeletal development pathways and ESM1 involved in the angiogenesis pathway exhibited a correlation between the amount of secretion and efficacy. These results suggest that these secreted factors may prove useful for predicting PD sheet efficacy and may therefore contribute to hyaline cartilage regeneration via PD sheets.

Decellularized Tissue for Muscle Regeneration

Several acquired or congenital pathological conditions can affect skeletal muscle leading to volumetric muscle loss (VML), i.e., an irreversible loss of muscle mass and function. Decellularized tissues are natural scaffolds derived from tissues or organs, in which the cellular and nuclear contents are eliminated, but the tridimensional (3D) structure and composition of the extracellular matrix (ECM) are preserved. Such scaffolds retain biological activity, are biocompatible and do not show immune rejection upon allogeneic or xenogeneic transplantation. An increase number of reports suggest that decellularized tissues/organs are promising candidates for clinical application in patients affected by VML. Here we explore the different strategies used to generate decellularized matrix and their therapeutic outcome when applied to treat VML conditions, both in patients and in animal models. The wide variety of VML models, source of tissue and methods of decellularization have led to discrepant results. Our review study evaluates the biological and clinical significance of reported studies, with the final aim to clarify the main aspects that should be taken into consideration for the future application of decellularized tissues in the treatment of VML conditions.

Xenogeneic Mesenchymal Stem Cells in the Formation of Hyaline Cartilage in Osteochondral Goat Failure

Background: Osteochondral knee failures are among the most common causes of disability among the elderly human population and animal athletes. The xenogeneic transplantation of mesenchymal stem cells is a questionable therapeutic alternative that, despite the low expression of Major Histocompatibility Complex type II by these cells, still has relevantuncertainties about the safety and clinical efficacy. The main objective of the present study was to investigate whether the xenogeneic transplantation of mesenchymal stem cells induces hyaline cartilage formation, without histopathological evidence of rejection, in osteochondralfailures of goats.Materials, Methods & Results: Five female goats were used, submitted to three surgical osteocondral failures in the right knee, treated with xenogenic mesenchymal stem cells of dental pulp, xenogenic platelet-rich plasma and hemostatic sponge of hydrolyzed collagen, respectively. The lesions were evaluated after 60 days of treatment, aiming to identify thepresence of hyaline cartilage or fibrocartilage and the subchondral bone pattern (regenerated or disorganized). Transplantation of xenogenic mesenchymal stem cells induced predominant formation of hyaline cartilage (P < 0.05), with no histopathological evidence of inflammationwhen compared to the other treatments. Therapies with xenogeneic platelet-rich plasma and hemostatic sponge of hydrolyzed collagen induced greater formation of fibrocartilaginous cartilage, with no significant difference between them (P > 0.05). Macroscopically, the lesions of the stem cell treated group showed formation of firm repair tissue, opaque staining, integrated with adjacent cartilage and with the failure filling almost completely. The groups treated with PRP and hemostatic sponge of hydrolyzed collagen presented, on average, partial filling of the lesion, with irregular shape and darkened coloration.Discussion. The absence of macroscopic and histopathological evidences of an inflammatory process on the surface and in the internal portion of the osteochondral lesions treated with xenogeneic stem cells, probably due to the low expression of Major Histocompatibility Complex type II by these cells, which would theoretically induce low rejection response. Such observations are of great importance, since graft-versus- host disease syndrome is a serious condition, responsible for the low therapeutic efficacy with transplantation of cells or grafts in humans. The formation of fibrocartilage, although without macro and microscopic evidence of degeneration or necrosis, in the osteochondral failures treated with PRP and hemostatic collagen sponge suggest that paracrine factors of the local microenvironment of the osteochondral failure are possibly responsible for the formation of fibrocartilaginous tissue or by inhibition of normal cartilage formation. The fibrocartilage formed in the Plasmaand Control groups, contributed to the commitment in the filling of the lesion, contrasting with the almost complete fill of the lesions treated with stem cells. The xenotransplantation of mesenchymal stem cells induced formation of hyaline cartilage and did not promote histopathological evidence of rejection in osteochondral lesions of goat knees. The treatments with PRP and hemostatic sponge of hydrolyzed collagen induced greater formation of fibrocartilaginous cartilaginous surface in the osteochondral failures.